Autocatalytic Formation of Green Heme:  Evidence for H₂O₂-Dependent Formation of a Covalent Methionine−Heme Linkage in Ascorbate Peroxidase

Abstract

The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the Cbeta of the 2-vinyl group to a methionine residue. To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant). The S160M variant is isolated from Escherichia coli as apo-protein. Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (kcat = 156 +/- 7 s(-1), KM = 102 +/- 15 microM) properties that are analogous to those of rpAPX. The reaction of S160M(R) with H2O2 gives a green protein (S160M(G)). Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160M(G). Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate. The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation. A mechanism that is consistent with the data is presented.

Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase

“…This was in direct contrast with the S160M variant, which gave a covalent haem adduct under the same oxidative conditions and timescales [10].…”

“…This haem peak now coincided with the protein peak (monitored at 280 nm) ( Figure 3B). Co-elution of haem and protein is clear evidence for the presence of a covalent haem-protein interaction and has been used previously for characterization of other covalently linked haem proteins [3,10,[18][19][20][21]. There was some protein degradation observed upon reduction of ferric S160C, resulting in the appearance of the broad peak eluted at 27 min in the 280 nm chromatogram ( Figure 3B).…”

“…The mutation was confirmed by sequencing across the entire rpAPX-coding gene. Plasmids containing the desired mutation were transformed into the E. coli BL21 (DE3) expression strain (Stratagene), and protein expression was carried out as reported previously [10].…”

“…The UV-visible spectrum (100 mM phosphate, pH 7.0, 25 • C) of ferric S160C (λ max = 414, 535 and 559 sh nm) (Figure 2A) showed increased formation of low-spin haem compared with rpAPX (λ max = 406.5, 504 and 634.5 nm [10]). …”

“…The second development has been the demonstration that the introduction of a methionine residue close to the 2-vinyl group in an S160M variant of rpAPX (recombinant pea cytosolic ascorbate peroxidase) leads to the formation of a vinyl-methionine link, analogous to that found in human myeloperoxidase, by means of an oxidized Compound I intermediate [10]. These results provide the first direct evidence that the formation of a vinylmethionine covalent linkage occurs as an H 2 O 2 -dependent (oxidative) process.…”